Atomic Level Analysis of Biomolecular Structure The overall objective of this proposal is to develop enhanced methods for analyzing structures of biological macromolecules at an atomic level of resolution. We build on our recent accomplishments and worldwide experience showing that single- and multi-wavelength anomalous diffraction (SAD and MAD) now predominate for de novo determinations of macromolecular crystal structures. We also build on the evolution of our own efforts in high-resolution structure determination to include analyses by cryogenic electron microscopy (cryo-EM). Our philosophy is to drive the development of methods by the demands of compelling applications to current problems of biological significance. We propose to optimize SAD and MAD phasing procedures and to enhance cryo-EM analyses of biomolecules at the atomic level in the course of studies of membrane proteins, macromolecular assemblages, eukaryotic proteins and other challenges. The overall objective is embodied in three specific aims: (1) We propose to enhance anomalous diffraction phasing procedures for challenging problems. One focus is on improved methods for increasing signal-to-noise ratios for anomalous diffraction by combining data from many crystals. A second focus is on enhanced MAD procedures for accurate experimental phase evaluation. (2) We propose to develop methods for the manipulation and diffraction analysis of microcrystals. One focus is on intrinsically small crystals, such as those of membrane proteins grown in lipidic cubic phase; and a second focus is on microcrystals purposed for the minimization of absorption, as is needed for low-energy native SAD experiments, or for rapid diffusion in time-resolved experiments. (3) We propose to enhance methods for analyzing cryo-EM structures using experience gained in analyzing x-ray crystal structures and in validating the fittings of atomic model interpretations to experimental EM maps.